Demembranated sea urchin sperm can be reactivated (induced to swim) with nearly normal wave geometry simply by adding exogenous ATP. The flagellar axoneme is known to contain at least two, and possibly more, species of ATPase activity. These observations suggest that ATP acts at several sites in the axoneme, not only to generate active microtubule sliding, but also to maintain the geometrically and temporally precise pattern of flagellar bending. The objective of this Proposal is to elucidate the structures and functions of ATP-dependent components in the axoneme by using drugs to poison specific parameters of flagellar motility. The detailed effects of motility-blocking drugs, including vanadate, erythro-9-(3-2(hydroxynony1)( adenine (EHNA), gossypol and adenosine-5'-0-(3-thiotriphosphate) (ATP-Gamma-S) on the beat frequency, wave forn, percentage motility, ATPase activity and energy coupling efficiency of demembranated, reactivated sea urchin spern will be investigated using dark-field microscopy, stroboscopy and microcinemtography. Dynein ATPases will be purified from sea urchin sperm tails, primarily by affinity chromatographic techniques, and the subunit structures of the purified enzymes will be characterized extensively using one- and two-dimensional SDS gel electrophoresis. One particular aim of the structural characterization is to identify the subunit containing the ATPase active site by tagging it with the photoaffinity label (Alpha-32P)N3ATP and identifying the (Alpha-32P)ATP-ATPase active site subunit complex by autoradiography of SDS polyacrylamidegels. Purified flagellar ATPases will also be tested for their responses to motility-blocking drugs with the goal of identifying each purified enzyme activity with a specific motility parameter. Drawing on the disciplines of biochemistry, cell biology and Pharmacology, the proposed studies should broaden our understanding of how ATP interacts with a variety of flagellar ATPases and, possibly, protein kinases, to produce the spatially and temporally controlled pattern of flagellar beating. Health-related issued addressed by this work include the molecular basis of flagellar and ciliary dysfunction and the pharmacological control of male fertility.